The overall aim of this proposal is to elucidate the mechanisms by which age-related insulin resistance and the metabolic syndrome promote atherogenesis. The specific research proposed in this application focuses on the hypothesis that increased free fatty acid (FFA) flux associated with age-related insulin resistance and the metabolic syndrome promotes atherogenesis through activation of the hexosamine pathway via mitochondrial overproduction of superoxide, and that this activation of the hexosamine pathway decreases endothelial nitric oxide synthase (eNOS) activity and increases plasminogen activator inhibitor- 1 (PAl- 1) transcription. Specific Aim 1 is to determine the effect of FFA-induced mitochondrial superoxide overproduction in bovine aortic endothelial cells (BAEC) on hexosamine pathway activation, eNOS modification by O-linked N-acetylglucosamine (GlcNAc) and phosphorylation, and eNOS activity, using adenoviral vectors that overexpress either uncoupling protein- 1 (UCP- 1) or manganese superoxide dismutase (MnSOD), antisense oligonucleotides for glutamine:fructose-6-phosphate amidotransferase (GFAT), UDP-GlcNAc measurements by HPLC, IP-Westerns for GlcNAc and phospho-eNOS, and an eNOS assay. Specific Aim 2 is to determine the effect of FFA-induced mitochondrial superoxide overproduction and hexosamine pathway activation on Sp 1 modification and PAI-1 promoter activity. BAEC will be treated with UCP-1, MnSOD, and GFAT antisense. Sp1 modification by GlcNAc and phosphorylation and Sp 1-dependent PAl-1 promoter activity will be determined, using IP-Westerns and luciferase reporter constructs. Specific Aim 3 will examine the effect of increased FFA flux on eNOS activity and PAl-1 expression in vivo in aortas of young and aging Sprague-Dawley rats using Liposyn infusion, with and without leptin, during euglycemic insulin clamps, eNOS will be evaluated by IP-Westems and activity assay, and PAl- 1 mRNA will be determined by real-time PCR and PAl-1 protein by a modified ELISA. Specific Aim 4 will examine the effects of a lipid-soluble thiamine derivative (benfotiamine) on FFA-induced hexosamine pathway activation, eNOS modification and activity, Spl modification and PAl-1 expression, both in BAECs and aortas of aging Sprague-Dawley rats. BAECs will be infected with an adenoviral vector expressing transketolase antisense to determine the mechanism ofbefotiamine action.